U.S. patent number 4,394,149 [Application Number 06/149,516] was granted by the patent office on 1983-07-19 for plant nutriment compositions and method of their application.
Invention is credited to Demetrios P. Papahadjopoulos, Francis C. Szoka, Jr..
United States Patent |
4,394,149 |
Szoka, Jr. , et al. |
July 19, 1983 |
Plant nutriment compositions and method of their application
Abstract
The disclosure is of the use of plant nutriments encapsulated in
synthetic lipid vesicles to nourish plants. SP CROSS-REFERENCE TO
RELATED APPLICATION This application is a Continuation-in-Part of
our copending application Ser. No. 881,116 filed Feb. 24, 1978 and
now U.S. Pat. No. 4,235,871.
Inventors: |
Szoka, Jr.; Francis C.
(Waltham, MA), Papahadjopoulos; Demetrios P. (Lafayette,
CA) |
Family
ID: |
26846806 |
Appl.
No.: |
06/149,516 |
Filed: |
May 13, 1980 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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881116 |
Feb 24, 1978 |
4235871 |
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Current U.S.
Class: |
71/28; 71/27;
424/60; 71/DIG.2; 71/64.11; 427/213.3 |
Current CPC
Class: |
A61K
9/127 (20130101); C05G 5/27 (20200201); C05C
9/005 (20130101); Y10S 71/02 (20130101) |
Current International
Class: |
A61K
9/127 (20060101); C05C 9/00 (20060101); C05C
009/00 () |
Field of
Search: |
;71/64F,27,28,64.11,DIG.2 ;210/500.2 ;424/19,38,60,319,365
;252/316 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Lurquin, 1979, Transfer of Plasmid DNA to Protoplasts Arch. Int.
Physiol. Biochim. 87(4) 825-826, (Abstract). .
Lurquin, 1981, Quamtitative . . . Protoplasts FEBS Lett., 125(2)
183-187 (Abstract). .
Lurquin, 1981, Binding . . . Exogenous DNA, Plant Sci. Lett., 21(1)
31-40 (Abstract). .
Rollo et al., 1981, Liposome . . . Analysis, Plant Sci. Lett.
20(4), 347-354 (Abstract)..
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Primary Examiner: Lander; Ferris H.
Attorney, Agent or Firm: Kane, Dalsimer, Kane, Sullivan
& Kurucz
Claims
What is claimed is:
1. A method of providing nutriments to agricultural plants
possessing plant cells having cell walls and cell membranes, which
comprises; applying to the plant a nutritional amount of a plant
nutriment, encapsulated in a lipid vesicle, whereby the lipid
vesicle encapsulating the plant nutriment is taken up by the plant
and passes into the plant cell where the lipid vesicle is broken
down and releases the encapsulated plant nutriment.
2. The method of claim 1 wherein the lipid vesicles are in a
dispersible form with a dispersible agriculturally acceptable
carrier.
3. The method of claim 1 wherein said applying is by spray.
4. The method of claim 1 wherein the nutriment is urea.
5. The method of claim 1 wherein the nutriment comprises ferrous
ethylenediamine tetraacetic acid complex and zinc ethylenediamine
tetraacetic acid complex.
6. The method of claim 1 wherein the nutriment is a fertilizer.
7. The method of claim 1 wherein the lipid vesicles have an average
diameter of from 2000 to 4000 angstroms.
8. The method of claim 7 wherein the lipid vesicles are
oligolamellar lipid vesicles.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to plant nutriments encapsulated in synthetic
lipid vesicles and to their use in agriculture to nourish
plants.
2. Brief Description of the Prior Art
Plants, like animals, are nourished by certain nutriments in order
to sustain their life. However, unlike animals, plants themselves
manufacture the organic nutriments required for assimilation into
protoplasmic materials, from more basic elements such as carbon,
hydrogen and oxygen. More specifically the plant will itself
manufacture the starches and sugars it requires for plant
metabolism from carbon, hydrogen and oxygen taken into the plant
structure. In addition to carbon, hydrogen, and oxygen, thirteen
other elements have been identified as essential to plant
nutrition. These are nitrogen and phosphorus (for protein
synthesis), potassium calcium, magnesium, sulfur, iron, manganese,
copper, zinc, boron, molybdenum, and chlorine. Although the latter
elements may be taken into the plant body through the leaf
structure, they are more usually taken in by the plant in the form
of minerals, dissolved in water and absorbed through the plant's
root system from the surrounding soil. These minerals as nutriments
may or may not be found in given soils. When they are not, the
agriculturist may resort to applying nutriment supplying
compositions to the plant situs in the form of fertilizer
compositions.
Plant nutrient or nutriment compositions such as commerical
fertilizer compositions are of various types. Most of them contain
water-disperable or water-soluble materials which can be
assimilated by plants. Fertilizers are representative of such
nutrient materials. The water-soluble ingredients, such as
nitrogen, potassium and phosphorus compounds which are the most
commonly used, are often too rapidly leached out of the fertilizer
composition are carried away by surface and ground waters long
before growing plants can gather and assimilate the nutritive
elements. Hence, in many cases a large proportion of desirable
nutritive elements is lost and to this extent the fertilizer or
other nutrient is wasted.
Numerous attempts have been made in the prior art to reduce water
solubility and the leaching rate of fertilizer and like nutrient
materials, while at the same time not rendering them incapable of
assimilation in growing plants. For example, a number of attempts
have been made to incorporate small quantities of fertilizer in
fairly high proportions of relatively water-insoluble carriers such
as asphalt, resins, plastics, wax and the like; see for example
U.S. Pat. Nos. 3,712,867; 3,778,383; and 3,321,298. In some cases
the fertilizer ingredients themselves have purposely been made
relatively insoluble. In other instances, granules or other small
particles of the fertilizer have been coated with water repellent
or water resistant materials including such as those named above.
In general, these procedures have not been very effective. Thus, if
the fertilizer particles are very highly waterproofed, they prevent
assimilation by the plants of the needed fertilizer ingredients. If
plastic hydrocarbon coating materials which retain a tacky or cold
flow property, such as wax or asphalt, are employed, they tend to
cohere and agglomerate unduly or to pack in large lumps during
periods of storage.
Other difficulties that are encountered in applying fertilizers and
like nutrients to soil are (a) neutralization of the materials by
reaction with soil components, e.g., phosphate fixation, and (b)
their destruction by microorganisms before they can be assimilated
by the plant, e.g., the action of denitrifying bacteria.
Basic to the method of our invention is the use of plant nutriments
encapsulated in lipid vesicles. When applied to a plant situs the
encapsulated nutriments are released slowly at the plant situs over
a period of time for absorption by the plant or they may be taken
in by the plant in their encapsulated forms. The encapsulated
nutriments remain fully bio-available for systemic plant nutrition
when absorbed into the plant structure and this unique delivery of
nutriments within the plant system may be advantageous in terms of
nutritional efficiency.
SUMMARY OF THE INVENTION
The invention comprises a method of nourishing plants, which
comprises; applying to the plant situs a nutritional amount of a
plant nutriment encapsulated in a lipid vesicle.
The terms "plant nutrient" and "plant nutriment" are
interchangeable and as used herein means a nutritious material for
plants, i.e.; one which has a nutrient or nourishing effect on the
plant.
The term "lipid vesicle" as used throughout the specification and
claims means a man-made (synthetic) liposome.
The term "oligolamellar lipid vesicles" as used herein means lipid
vesicles as previously defined and characterized in part by few or
single bimolecular lipid layers forming the vesicle walls.
The term "plant situs" as used herein means the environmental zone
surrounding a given plant, which ordinarily provides nutritional
material to the plant.
The method of the invention employs the lipid vesicles containing
nutriments for sustained slow release, to effect the growth of
plants.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
Prior to our invention, several methods were available to make
synthetic liposomes, encapsulating biologically active materials;
see for example Bangham et al. in J. Mol. Biol., 13:238-252 (1965);
D. Papahadjopoulos and N. Miller (Biochim. Biophys. Acta,
135:624-638 [1967]); Batzri and Korn (Biochim. Biophys. Acta,
298:1015 [1973]); Deamer and Bangham in Biochim. Biophys. Acta,
443:629-634 (1976); Papahadjopoulos et al. in Biochim. Biophys.
Acta, 394:483-491 (1975); German Pat. No. 2,532,317; and U.S. Pat.
Nos. 3,804,776 and 4,016,100.
The prior art methods of making synthetic liposomes may be employed
to encapsulate plant nutriments for use in the method of our
present invention. However, in a preferred embodiment of our
invention the lipid vesicles encapsulating plant nutrients are
prepared by our new method of lipid vesicle preparation.
By our new method, oligolamellar lipid vesicles (synthetic
liposomes) may be constructed rapidly, conveniently, under mild
conditions, in high yields, and in such a manner that they
incorporate a high percentage of a wide variety of plant nutriments
processed with them. Thus, there is an economic advantage to the
encapsulation of plant nutriments by the preferred method.
The preferred method of plant nutriment encapsulation may be
carried out by first providing a mixture of a vesicle wall forming
compound in organic solvent and an aqueous mixture of the nutrient
material to be encapsulated, the ratio of organic phase to aqueous
phase being that which will produce an emulsion of the water-in-oil
type. One then forms a homogeneous emulsion of said mixture, of the
character produced by ultra-sonic radiation. By removing organic
solvent from the emulsion, a mixture is obtained having a gel-like
character. Then by converting the gel-like mixture to synthetic,
oligolamellar vesicles one encapsulates the nutrient material.
The invention also comprises the intermediate gel-like material,
the product synthetic lipid vesicles encapsulating nutriments,
their use and agriculturally acceptable carrier compositions
including the synthetic vesicles as the active ingredient
thereof.
In a broad sense, the above-described preferred preparation of the
lipid vesicles calls for the formation first of "inverted micelles"
in an organic phase and then the removal of the organic phase. The
system then spontaneously reverts to a bilayer-like structure, with
a large amount of aqueous phase encapsulated in large oligolamellar
vesicles. The advantage of this method is that it gives high
capture efficiencies of nutriment containing aqueous phase and
provides large, stable vesicles. Phospholipids are excellent
molecules for the formation of the "inverted micelles" and then the
subsequent bilayer of the vesicles. More specifically, the method
of the lipid vesicle preparation may be carried out as follows.
The first step is to provide a mixture of a lipid vesicle wall
forming composition in organic solvent and an aqueous mixture of
the plant nutriment to be encapsulated in the vesicle. Vesicle wall
forming compounds are generally well known as are the methods of
their preparation. For example, any number of phospholipids or
lipid compounds may be used to form the vesicle walls.
Representative of such wall forming compounds are:
phosphatidylcholine (hereinafter referred to as "PC"), both
naturally occurring and synthetically prepared, phosphatidic acid
(hereinafter referred to as "PA"), lysophosphatidylcholine,
phosphatidylserine (hereinafter referred to as "PS"),
phosphatidylethanolamine (hereinafter referred to as "PE"),
sphingolipids, phosphatidyglycerol (hereinafter referred to as
"PG"), spingomyelin, cardiolipin, glycolipids, gangliosides,
cerebrosides and the like used either singularly or intermixed such
as in soybean phospholipids (Asolectin, Associated Concentrates).
In addition, other lipids such as steroids, cholesterol, aliphatic
amines such as long chain aliphatic amines and carboxylic acids,
long chain sulfates and phosphates, dicetyl phosphate, butylated
hydroxytoluene, tocophenol, retinol, and isoprenoid compounds may
be intermixed with the phospholipid components to confer certain
desired and known properties on the formed vesicles. In addition,
synthetic phospholipids containing either altered aliphatic
portions such as hydroxyl groups, branched carbon chains,
cycloderivatives, aromatic derivatives, ethers amides,
polyunsaturated derivatives, halogenated derivatives or altered
hydrophillic portions containing carbohydrate, glycol, phosphate,
phosphonate, quaternary amine, sulfate sulfonate, carboxy, amine,
sulfhydryl, imidazole groups and combinations of such groups can be
either substituted or intermixed with the above mentioned
phospholipids and used in the preferred process of lipid vesicle
formation. It will be appreciated from the above that the chemical
composition of the lipid component of the vesicles prepared may be
varied greatly without appreciable diminution of percentage
nutriment capture although the size of the vesicle may be affected
by the lipid composition. A convenient mixture we have used
extensively and which is representative of lipid mixtures
advantageously used in preferred lipid vesicle preparation is
composed of PS and PC, or PG and PC as identified above
(advantageously at a 1:4 molar ratio in each instance). The PC, PG,
PA and PE, may be derived from purified egg yolk. Saturated
synthetic PC and PG, such as dipalmitoyl may also be used. Other
amphipathic lipids that may be used, advantageously in various
mixtures, are gangliosides, globosides, fatty acids, stearylamine,
long chain alcohols, long chain sulfates and the like.
The liposome wall forming composition may be initially provided
dissolved in any inert solvent that can be substantially removed
from the lipid or phospholipid compound when desired.
Representative of such solvents are a wide variety of ethers,
esters, alcohols, ketones, hydrocarbons (aromatic and aliphatic
including fluorocarbons), and silicones in which an aqueous phase
does not have an appreciable solubility. The solvents may be used
either alone or in admixture. For each solvent or mixture of
solvents however, the optimal ratio of lipid, aqueous space, and
solvent is different and must be determined for each case by trial
and error techniques as will be appreciated by those skilled in the
art. The term "inert solvent" as used herein means a solvent for
the lipid or phospholipid, which will not interfere with or
otherwise adversely affect the desired course of the preferred
preparative method.
The phospholipid or lipid along with any lipid-soluble additives,
are advantageously evaporated from their solvent on to the sides of
a suitable reaction vessel. The organic phase, in which the
"reversed phase evaporation vesicles" will be formed is then added
to the vessel, i.e.; an inert organic solvent for the lipids and
phospholipids as described above. With mixing, dissolution of the
lipid component of the vesicles to be formed, previously deposited
on the vessel walls is obtained. A number of inert organic solvents
are preferred for forming the organic phase according to the
preferred preparative method, depending on the following conditions
employed. For low temperature conditions, i.e.; removal
subsequently of the organic phase at relatively low temperatures,
we find diethyl ether most advantageous, although chloroform, or
tetrahydrofuran may also be used advantageously. For higher
temperature processing, isopropyl ether is a preferred inert
organic solvent, particularly for preparing lipid vesicles
containing saturated phospholipids as the lipid component.
Following dissolution of the phospholipid or lipid to form the
organic phase, an aqueous phase is added to obtain a heterogeneous
2-phase mixture. The aqueous phase contains in
dissolution/suspension the nutriment compounds or compositions to
be encapsulated in the synthetic lipid vesicles produced by the
preferred preparative method. Preferably the aqueous phase is
buffered to a pH suitable to maintain stability of the material for
encapsulation. The ionic strength of the aqueous phase has a
bearing on the encapsulation efficiency obtained in the preferred
preparative method. As a general rule, the higher the ionic
strength of the aqueous phase, the lower the percentage of
entrapment. For example, with 15 mM sodium chloride present, one
can encapsulate circa 60 percent of the aqueous phase, while with
500 mM sodium chloride present, only about 20 percent of the
aqueous phase may be encapsulated. Thus, to maximize the
encapsulation of macromolecules, a buffer of low ionic strength
(less than 0.3) is preferably employed. The encapsulation
efficiency is also dependent to some degree on the concentration of
lipid or phospholipid present in the 2-phase system. Preferably the
proportion of lipid or phospholipid component is within the range
of from about 0.5 mg to about 50 mg/ml. of the inert organic
solvent. Preferably the ratio of organic phase to aqueous phase is
within the range of from about 2:1 to about 20:1 v/v, most
preferably about 4:1 to form a water-in-oil emulsion.
The heterogeneous 2-phase mixture obtained as described above is
then emulsified to obtain an emulsion of the character produced by
ultrasonic radiation. Preferably this is accomplished with the use
of a bath type sonicator, or for large volume preparations in an
industrial size emulsifier. Generally, the 2-phase mixture is
sonicated for about 3 to 5 minutes, or until either a clear 1-phase
mixture or a homogeneous emulsion forms. This is achieved by simply
placing the container vessel in the sonicating bath at an optimal
level. Emulsification may be carried out over a wide range of
temperatures, i.e.; from about -10.degree. to about 50.degree. C.,
advantageously at a temperature of from 0.degree.-20.degree. C. The
optimum conditions under which emulsification is carried out
depends upon the solvent, phospholipid, and volume of aqueous phase
used in the preparation. It will be appreciated that trial and
error techniques may be used to determine the optimum conditions
for emulsification. The emulsion mixture is then treated to remove
a substantial portion of the inert organic solvent. This may be
carried out conveniently by use of a rotary evaporator, at a
temperature of circa 20.degree. C. to 60.degree. C. and under a
reduced pressure, i.e.; under vacuum (10 mm to 50 mm Hg). The
temperature employed for evaporation of the organic solvent from
the emulsion depends on the boiling point of the particular organic
solvent used in the emulsion and the stability of the nutriment
material being encapsulated. During evaporation, the emulsion first
becomes a viscous gel, which is an intermediate product. The gel is
stable and can be stored in this state for short periods of time,
up to a week (at least), at 4.degree. C. under an inert atmosphere
such as nitrogen gas. A small amount of water or buffer can then be
added to the gel and the resulting mixture evaporated for an
additional period (circa 15 minutes) to help remove residual traces
of the organic solvent, and to speed the conversion of the gel into
a homogeneous-appearing, suspension of oligolamellar lipid
vesicles. The gel may be converted by agitation or by dispersion in
an aqueous media such as a buffer solution. The vesicles obtained
range in diameter from 2,000 to 4,000 angstroms (average). A
significant proportion of the nutriment compounds for encapsulation
contained in the aqueous buffer is captured within the lipid
vesicles (up to circa 60 percent, depending on the amount of lipid,
volume of the aqueous phase, ratio of the organic phase to aqueous
phase to lipid, type of inert organic solvent(s) and, type of
lipid(s) used in the process). The non-incorporated aqueous
material may be removed if necessary by appropriate and known
techniques such as by repeated centrifugations, column
chromatography, ion exchange chromatography, dialysis and like
procedures. Generally however separation is not necessary and the
crude mixture may be used as is in the method of the invention. The
lipid vesicles with their encapsulated contents may be suspended in
any isotonic buffer for use. The vesicles may be sterilized by
passage through a 0.4 micron filter (nucleopore) when sterility is
desired.
Advantageously the preferred method of preparing nutrient
containing lipid vesicles is carried out under an inert atmosphere.
The term "inert atmosphere" as used herein means a non-oxidizing
atmosphere such as an atmosphere of nitrogen gas, argon and like
inert gases.
Representative of nutriment materials that may be encapsulated by
the method described above are vitamins, minerals, growth
stimulating hormones, growth promoters such as diallyl pimelate,
the auxins such as 3-indoleacetic acid, 3-indolebutyric acid and
the like, the gibberellins such as gibberellic acid (GA.sub.3) and
the like; fertilizers such as urea, calcium cyanamide, ammonium
nitrate, ammonium sulfate, sodium nitrate, calcium nitrate,
ammonium phosphate, potassium nitrate, potassium chloride, mixtures
thereof (multinutrient fertilizers) and the like. The encapsulated
nutriments cannot be readily removed from the area site of
application (plant situs) by rain or irrigation. Encapsulation of
the nutrient materials protects them from inactivation or removal,
i.e.; maintains plant bioavailability for agricultural crops to
which the encapsulated material is applied according to the method
of our invention.
It will be observed from the above that our preferred method of
preparing nutrient containing lipid vesicles differs from the prior
art methods, of making vesicles in several ways. For example,
according to our preferred method the nutrient material to be
encapsulated is added into the organic phase with the lipid where
it is totally encapsulated. Furthermore, the organic phase is
substantially removed before an excess of an aqueous phase is
added. The emulsification of the initial aqueous phase into the
organic phase, and the removal of the organic phase prior to the
addition of any excess aqueous phase is essential for high capture
percentage in this method and is a critical difference between our
preferred process and all previous methods heretofore described for
lipid vesicle preparation. The method we advocate produces large
oligolamellar vesicles from many different lipids either alone or
in combinations. A further advantage is that the evaporation of the
organic phase is performed under mild temperatures and vacuum to
obviate the potential for inactivation of sensitive nutrient
molecules.
The method of the invention is carried out by applying to a plant
situs, a nutritional amount of a plant nutrient encapsulated in a
lipid vesicle. Application may be to the plant leaves, but is
preferably to the surrounding soil. The amount which constitutes a
nutritional amount will vary depending on the plant, plant
nutritional needs, etc.
The question in most instances is how much fertilizer to use on a
soil that is not at an optimum level of fertility. Soil tests can
be made by which the amount of available nutrients in the soil can
be determined. The amounts needed for various plants and crops are
generally known. From experience it can then be predicted how much
fertilizer or nutriment should be applied to a given situs, i.e.;
the rate of nutriment application. Those skilled in the art of
agriculture will appreciate how to determine rates of nutriment
application.
The lipid vesicle encapsulated nutriment compositions may be
applied to a given plant situs in a dispersible pure form, i.e.; as
obtained directly by the above-described method of encapsulation
but dispersible agricultural formulations for fertilizer uses are
preferred. The dispersible agricultural formulations of this
invention comprise a lipid vesicle encapsulated nutriment in a
homogeneous, dispersible form with a homogeneous, dispersible,
agriculturally acceptable carrier. A homogeneous, dispersible,
agriculturally acceptable carrier preferably comprehends a liquid
carrier diluent. The lipid vesicles can be dispersed in a liquid
carrier diluent as finely divided particles (suspension).
The term "dispersible", as used in this specification and in the
claims, means matter dispersed in a liquid such that it can be
evenly distributed over a given area or situs. Emulsions and
suspensions of lipid vesicles in water are one embodiment of
dispersible formulation contemplated.
In addition to containing the nutriment encapsulated lipid
vesicles, the dispersible carrier formulations of the invention may
include a variety of adjuvants such as humecants, dispersants,
adhesive or sticking agents, corrosion inhibitors, and anti-foaming
agents. Illustrative of such adjuvants are humecants such as
glycerol, diethylene glycol. solubilized lignins (such as calcium
ligninsulfonate) and the like. Dispersants include methyl
cellulose, polyvinyl alcohol, sodium ligninsulfonate and the like.
Adhesive or sticking agents include vegetable oils, naturally
occurring gums, casein, and the like. A representative corrosion
inhibitor is epichlorohydrin, and a representative anti-foaming
agent is stearic acid. The concentration of the active ingredient,
i.e.; the lipid vesicles present in the agriculturally acceptable
carrier is not critical and any concentration desired may be used.
As a practical matter, from 10 to 80 percent by weight lipid
vesicles in the carrier are advantageous concentrations.
The most important factor is how much nutriment is applied to a
given plant situs. It is readily apparent that one can apply a
large amount of a formulation having a low concentration of
nutriment or a relatively small amount of a formulation having a
high concentration. Whether a low or high concentration should be
used depends upon the mode of application, the amount and kinds of
vegetation, and the thoroughness of coverage desired. The total
amount to be applied depends upon the kinds of crop, the severity
of nutritional need, the state of plant development, and the season
of the year as those skilled in the art will appreciate and as
discussed above.
Representative homogeneous dispersible formulations according to
this invention include sprays and delivery by irrigation means such
as canals, etc. Spray formulations are preferred for foliar
applications and for uniformly controlled applications to a soil.
The spray formulations in accordance with the invention may be
aqueous suspensions, oil-in-water emulsions and the like. The lipid
vesicles may also be dispersed in a suitable water-miscible inert
organic liquid. Representative of water-miscible, inert organic
liquids are acetone, methyl ethyl ketone, dimethylformamide,
alcohols, monoalkyl ethers of ethylene glycol, ethyl acetate, and
the like. The spray formulations will conveniently comprise from
about 0.1% or lower to about 50% by weight or even higher, a volume
of spray being applied so that a nutritionally effective amount of
nutriment is applied to a given situs.
The following examples describe the manner and process of making
and using the invention and represent the best mode contemplated by
the inventors, but are not to be construed as limiting. In all of
the procedures described below, one can include 0.5 to 1 mole of a
fluorescent phospholipid analog such as NBD-PE (Avanti
Biochemicals) with the lipid or phospholipid component in order to
be able to visually follow the separation of the vesicles on a
column.
EXAMPLE 1
Encapsulation of Urea
A 50 ml round bottom flask with a long extension neck is fitted
with a 24/40 fitting so as to conveniently couple to a flask
evaporator. The flask is also fitted for continuous purging with
nitrogen gas. The flask is charged with 300.mu. moles of
phospholipid-phosphatidylglycerol/phosphatidylcholine/cholesterol
(PG/PC/Chol) 1/4/5 molar ratio in 15 ml diethylether and 5 ml of a
solution of 8 M urea in water is added. The mixture is emulsified
by ultrasonic treatment. The organic solvent is then removed by
evaporation. The preparation so formed contains a mixture of 50%
encapsulated urea and 50% non-encapsulated urea that can be applied
to agricultural crops as a slow release fertilizer. Application may
be by spray technique. The encapsulated urea is protected from
washout by rain or irrigation and from degradation by urease
enzymatic activity in the soil. The lipid vesicles are also wholly
absorbed by the plant through its root structure and deliver the
nutriment urea at the cellular level.
EXAMPLE 2
Encapsulation of Micronutrients in Vesicles as a Slow Release
Fertilizer to Treat Plants Afflicted by Iron Chlorosis
Five ml of a solution containing 1% ferrous ethylene diamine
tetraacetic acid complex (EDTA) 1% zinc EDTA is added to the flask
described in Example 1, containing 15 ml of diethylether containing
300.mu. moles of PG/PC/Chol:1/4/5. Vesicles are prepared as
described in Example 1 above. The unencapsulated Fe-EDTA, and
ZnEDTA are removed by dialysis and the FeEDTA and ZN-EDTA that
remains encapsulated may be spray applied to agricultural plants as
a slow release micronutrient reservoir to treat chlorosis in
affected plants.
By changing the lipid composition from PG/PC/Chol to DSPC
(distearoylphosphatidylchloine) and encapsulating the above
compounds the rate of release of the fertilizer will be appreciably
slowed when compared to the PG/PC/Chol composition. Thus, by using
phospholipids with different characteristic transition
temperatures, one can prepare liposomes that will have defined
permeability rates. The shorter the acyl chains and the greater the
degree of unsaturation of the phospholipids used to prepare the
vesicles the faster the rate of leakage of the nutrients from the
vesicles. The lipid vesicles are taken into the plant system
through the root structure and assimilated within the plant, at the
cellular level.
* * * * *